Viral RNA testing and automation on the bead-based CBNE detection microsystem.

Galambos, Paul C. ; Bourdon, Christopher Jay ; Farrell, Cara M. ; Rossito, Paul (University of California at Davis) ; McClain, Jaime L. ; Derzon, Mark Steven ; Cullor, James Sterling (University of California at Davis) ; Rahimian, Kamayar

Sandia National Laboratories

关键词: Nucleic Acids;    Modifications;    Sensitivity;    Nucleic Acid Hybridization;    Diseases;   

DOI  :  10.2172/947327
RP-ID  :  SAND2008-7332
RP-ID  :  AC04-94AL85000
RP-ID  :  947327

美国|英语

来源: UNT Digital Library

【 摘 要 】

We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and immunoassay detection.